The long-term goal of this proposal is to understand the functions of H1 linker histone in chromosome structure and activity. We propose to use the fruit fly, Drosphila melanogaster, as a model system because it provides many advantages for studies of chromosome structure and genetic activity and because Drosophila H1 strongly resembles mammalian H1 linker histones. In metazoans, H1 is nearly as abundant as nucleosome core particles, suggesting that it plays important roles in establishing and maintaining the architecture of the chromatin fiber. H1 can influence chromatin functions at several levels, including locally at the level of individual nucleosome particles and arrays as well as over long range to determine the folding of chromatin into higher order structures. H1 is expected to affect gene expression and many other reactions of nuclear DNA metabolism, such as replication, recombination and repair. Much of our knowledge about the roles of linker histones comes from in vitro studies. However, in vivo analyses of H1 functions have been lagging. Recently, we used RNA interference (RNAi) to nearly completely deplete H1 in Drosophila melanogaster in vivo. This approach allowed us to demonstrate that H1 is essential for Drosophila development and that it is required for normal chromosome architecture. We identified heterochromatin structure and activity as primary targets of H1 regulation. We also found that H1 has both positive and negative effects on gene expression. To gain a deeper understanding of the mechanisms by which H1 controls chromosome structure and gene activity, to identify the factors that work in concert with H1 to regulate the epigenetic states of the genome, and to discover other cellular processes that depend upon H1 for proper function, we propose to pursue the following specific aims. (1) We will elucidate mechanisms by which H1 regulates the structure and activity of heterochromatin. (2) We will characterize the network of proteins that functionally interact with H1 in vivo. (3) We will perform a detailed in vivo structure-function analysis of H1. The successful completion of the proposed studies is expected to provide crucial insights into the ways in which this major component of chromatin affects the structure and activity of eukaryotic chromosomes. PUBLIC HEALTH RELEVANCE: The goals of this project are to understand the functions of a major component of all eukaryotic chromosomes called H1 linker histone. H1 is a key determinant of the structure and genetic activity of chromosomes. By collaborating with other components of chromosomes, H1 regulates the epigenetic state of the genome. Thus, H1 is a key intermediate in process that control normal embryonic/fetal development. When these processes are perturbed they contribute to the development of numerous human diseases including cancer.